Abnormal thalamocortical connectivity of preterm infants with elevated thyroid stimulating hormone identified with diffusion tensor imaging

While thyroid disturbances during perinatal and postnatal periods in preterm infants with congenital hypothyroidism reportedly disrupt neuronal development, no study has considered the effect of thyroid disturbances in premature infants with subclinical hypothyroidism with elevations of thyroid stimulating hormone. We aimed to identify altered fiber integrity from the thalamus to cortices in preterm infants with subclinical hypothyroidism. All preterm infants born were categorized according to thyroid stimulating hormone levels through serial thyroid function tests (36 preterm controls and 29 preterm infants with subclinical hypothyroidism). Diffusion tensor images were acquired to determine differences in thalamocortical fiber lengths between the groups, and cerebral asymmetries were investigated to observe neurodevelopmental changes. Thalamocortical fiber lengths in the subclinical hypothyroidism group were significantly reduced in the bilateral superior temporal gyrus, heschl’s gyrus, lingual gyrus, and calcarine cortex (all p < 0.05). According to the asymmetric value in the orbitofrontal regions, there is a left dominance in the subclinical hypothyroidism group contrary to the controls (p = 0.012), and that of the cuneus areas showed significant decreases in the subclinical hypothyroidism group (p = 0.035). These findings could reflect altered neurodevelopment, which could help treatment plans using biomarkers for subclinical hypothyroidism.


Results
We investigated a cohort of 106 preterm infants. After applying the exclusion criteria, a total of 27 infants were excluded: interventricular hemorrhage (n = 17), periventricular leukomalacia (n = 4), small for gestational age (n = 3), Down syndrome (n = 1), Russell-silver syndrome (n = 1), and maternal thyroid diseases (n = 1). In a cohort of 79 infants, we have classified infants with normal TSH levels (≥ 6; n = 35) and infants with abnormal TSH levels (< 6; n = 44) based on TSH levels within 6 weeks of life. Finally, after excluding 14 insufficient MR, the final cohort consisted of 29 preterm infants with SH and 36 preterm infants without SH (control group, CN) ( Fig. 1). Additionally, 2 of SH group exhibited received levothyroxine (Tx before discharge, n = 1; Tx after discharge, n = 1). Among the 2 infants who were treated with levothyroxine, 1 infant born at 27 weeks' gestation had normal results on the initial TSH and free T4 (fT4). Serum TSH levels gradually increased during follow-up, and at 6 weeks of life reached peak TSH levels (44 μU/mL) with normal fT4 levels (0.8 ng/mL). The other infant born at 26 weeks' gestation was started on levothyroxine treatment after discharge with persistently elevated serum TSH levels (between 10 and 19.9 μU/mL). The TSH levels averaged across the thyroid function tests (TFT) were significantly higher in the SH group (11.56 ± 8.12 μU/mL) than in the CN (3.65 ± 1.37 μU/mL; p = 0.001). The individual TFT at each time point differed significantly between the SH group and CN group at the first (SH: 7.31 ± 1.07 μU/mL, CN: 1.74 ± 1.23 μU/mL, p < 0.001) and second time points (SH: 11.07 ± 7.70 μU/mL, CN: 3.29 ± 1.53 μU/mL, p < 0.001), but not at the third (SH: 8.39 ± 6.46 μU/mL, CN: 3.57 ± 1.55 μU/mL, p = 0.007) ( Table 1). Furthermore, the individual fT4 levels were not significantly different from between the SH group www.nature.com/scientificreports/ The fiber lengths were measured to check the disturbances of the thalamocortical pathway in the SH. Compared to the healthy controls, infants with SH featured significant reductions in the fiber lengths in the cerebral regions of the SH. The bilateral abnormalities in fiber lengths included the superior temporal gyrus (STG), heschl's gyrus (HES), calcarine cortex (CAL), and lingual gyrus. Abnormalities found only in the right and left hemispheres included the right orbitofrontal (ORB) and the left middle frontal gyrus (MFG), rolandic operculum (ROL), middle cingulate gyrus (MCG), superior occipital gyrus (SOG), and cuneus (CUN), respectively. In both cerebral regions, fiber lengths were 62-73% shorter in infants with SH relative to their CN counterparts (HES, 71.6%; CAL, 70%; LING, 68.7%; and STG, 63.5%; Fig. 2), accounting for the significant differences between the two populations (p < 0.05). Significant reductions in fiber lengths of 49 Fig. 3). The fiber lengths are summarized in Table 2.   www.nature.com/scientificreports/ We also employed the lateralization index to assess hemispheric dominance in regions wherein the length of fiber tracts differed significantly between the SH and CN populations. The asymmetric values showed an overall left dominance, excepting the STG and CAL (Fig. 4). In the unilateral areas, while ORB was dominant in the right hemisphere of CN infants, it became left-dominant in the SH group (SH: 0.154 ± 0.129, CN: -0.270 ± 0.086, p = 0.012). In addition, the CUN was significantly lower in the SH group (0.395 ± 0.100) than in the CN group (0.703 ± 0.073; p = 0.035). The lateralization values in the other regions showed slight increases or decreases, but none were significant (Table 3).

Discussion
In this study, we employed DTI to quantify the fiber integrity of preterm infants with SH and thus determined the significant reductions in fiber lengths in the thalamocortical pathways of infants with SH in the following regions: the bilateral STG, HES, CAL, and LING; and the unilateral ORB, MCG, MFG, ROL, SOG, and CUN. In addition, compared to controls, the lateralization values of two cerebral regions significantly changed; the laterality of the ORB was transposed to the opposite hemisphere in infants with SH, and that of the CUN decreased significantly. These results indicate disrupted fiber connectivity of the thalamocortical pathway in the frontal, temporal, and occipital lobes of infants with SH, reflecting abnormal white matter myelination and impaired cortical growth in this clinical population. It is noteworthy that the fiber lengths in the SH group were significantly decreased in the temporal lobe, including the regions of the STG and HES, suggesting reductions of dendrite and synaptic extensions. TH regulate the generation and number of oligodendrocytes, which are related to the activation of subplate neurons that help to establish transient synaptic contact from the thalamus to the cortex [40][41][42] . The development of subplate neurons is prominent at mid-and late-fetal phases, and disturbances during these periods induce the reorganization of the cortical development in perinatal and early postnatal infants 43 . Several studies on CH have found cortical thinning in the temporal lobe of children and adults with regard to neuronal loss in the temporal lobe 36,37 . In rodent models of transient and CH, abnormal cellular integrity has also been observed using electron microscopy 24,25 , implicating TH in neuronal death. Moreover, adult-human connectome research has reported that short fibers are positively correlated with lower cortical thickness 44 , which supports our hypothesis that the reduced fiber lengths of preterm infants suffering from SH could be used as a biomarker to signal neuronal deficits in the temporal lobe, since the advent of the SH precedes thyroid dysfunction in preterm infants 7 . DTI could be a more sensitive marker for monitoring neurodevelopment than the morphometric analyses based on three-dimensional T1-weighted imaging 45 . However, further studies are needed to validate the relationship between fiber length and cortical thinning in preterm infants.
We also demonstrated significant bilateral fiber reductions in the occipital and frontal lobes (CAL and LING, Fig. 2) and the lobes that exhibited unilateral changes in fiber length (Fig. 3), and the lateralization states of the CUN and ORB in the frontal and occipital lobes, respectively, differed clearly between the two populations (Fig. 4). These phenomena could be expected to postpone normal neonate growth, causing abnormal neurodevelopment patterns. In the fetal stages, the thalamocortical pathway first progresses to the anterior and middle cerebral regions until 21 weeks of gestational age, matures posteriorly from 22 weeks of gestational age, and condenses and organizes the projection of specific cerebral cortex until 40 weeks of gestational age 17 . Interestingly, similar to our findings, a DTI study of infants born before 28 weeks with low T4 concentrations observed increased apparent diffusion coefficient values in the occipital region at term-equivalent ages, indicating poor white matter formation due to thyroid disturbance 39,45 . This finding further suggests that, in the context of the thyroid hormone-activated sensitive period of mid-and late-fetal development 43 , the abnormal thyroid state affects that the decreased fiber lengths in the SH could indicate the postponement of fiber aggregation in the occipital lobe. On the other hand, there are developmental differences in cerebral volume, sulci, and fiber connectivity in fetuses, preterm infants and full-term neonates [46][47][48] . The ORB volumes in healthy fetuses 48 , the lateral ORB network in full-term neonates 46 , and the frontal sulci in preterm infants 47 favor right hemispheric dominance across aging. In addition, the centrality of the CUN shows left dominance in neonates 46 , which is comparable to the lateralization features of preterm controls in this study. However, our preterm infants with the www.nature.com/scientificreports/ SH exhibited a different pattern of asymmetries in the CUN and ORB, compared to preterm controls (Fig. 4). This result indicates that the changed lateralization in the occipital and frontal lobes may represent neurodevelopmental disorders caused by defective fiber maturation. Consequently, we suggest that the decreased fiber lengths in the corresponding cerebral regions accompanying the lateralization changes would manifest as developmental delay due to the slow activation of the thyroid hormone. However, additional studies using DTI are required to investigate the longitudinal lateralization patterns of preterm infants with SH, as increased cortical thickness in the occipital and frontal regions in infants with the CH has been implicated in developmental deformation due to the lengthened maturation in adolescence consequent of CH 36 .
Most neuroimaging studies related to CH have identified abnormalities in various cerebral regions, including the frontal, temporal, and occipital lobes 36,37,39 , resulting in memory and visuospatial disorders in relation to cognitive function at school ages 36,[49][50][51] . These symptoms have also been found in children with constant thyroid disturbance during the neonatal stage 52 , which could be perceived as a valuable clinical warning for preterm infants with thyroid dysfunction. Importantly, the thyroid hormone should be more carefully monitored in the early birth, since 17-40% of newborns with abnormal TH levels develop CH 10 . However, thyroid symptoms are difficult to diagnose within a few weeks of birth because of the lack of remarkable clinical signs during this period 53 . We found a reduction in fiber length in the SH as a prodrome of thyroid pathophysiology using a non-invasive DTI approach, implying that it can be used as a reliable imaging biomarker to assist clinically efficient cures for the normal development of preterm infants. Furthermore, fiber abnormalities identified using DTI have been positively correlated with mental, cognitive, and motor function in preterm infants 35,54 . When combined with serial thyroid testing, fiber changes determined with DTI could be used as a quantitative clinical marker to help anticipate whether cerebral development appropriately progresses while preterm infants with SH undergo treatment.
This study is subject to several limitations that need to be addressed in future research. First, this study employed a single-center, retrospective, and uncontrolled observational design, and was additionally limited by variations in the timing and interval of TFT screening. Moreover, we did not account for other potential confounders, including maternal factors of iodine intake, drug use, the existence of thyroid disease or thyroid autoantibodies, and those that depended entirely on the decisions of the attending pediatric endocrinologists, including variations in the indication, timing, and duration of levothyroxine replacement therapy. We also note that the small number of infants subjected to MRI and DTI analyses was insufficient for the subgroup analysis of SH and transient hypothyroxinemia of prematurity. Furthermore, we did not monitor iodine exposure in infants, although we routinely used 0.5% chlorhexidine instead of iodine-containing agents as skin antiseptics during the procedures at our institute throughout this study period. As only 10% of patients underwent surgical procedures, the effect of iodine exposure on the incidence of TSH elevation might be insignificant. Regarding imaging analysis, recent DTI studies have been implemented with multi-shell imaging techniques with two or more different b-values to show fiber distributions of the white matter more clearly 55 . However, as the DTI dataset was acquired with a b-value of 1000 s/mm 2 and the number of 16 directions. These could cause a different sensitivity of the fiber lengths due to a variety of diffusion factors such as diffusion anisotropy, crossing fibers, and the number of axons in a voxel 56 , which can reduce the quality of tract reconstruction. Additional studies with high b-value and gradient directions can reinforce the outcomes of this study in the future. In addition, thalamic volume can affect the potential difference of fiber lengths of thalamocortical tract. The relationship between structure alteration and fiber integrity should be further explored by follow-up studies to consolidate the present study.
We investigated fiber lengths using DTI to identify abnormalities in the thalamocortical pathway in preterm infants with SH. Defective fiber integrity accompanied by lateralization changes were demonstrated in several cerebral regions, including the frontal, temporal, and occipital lobes, of infants with SH. These results could help to establish a reasonable plan for the treatment of thyroid dysfunction and thus prevent the onset of cognitive impairments related to memory and visuospatial disorders in toddlers, children, and adults.

Methods
Study populations. All preterm infants who were admitted to the neonatal intensive care unit of Hanyang University Clinic for Developmental Disorders were initially recruited to investigate their cerebral development between February 2016 and January 2019. Preterm birth was defined as being born before 32 weeks of gestation with a birth weight of < 1.5 kg. All recruited infants were confirmed through MRI and ultrasonography screenings to have no brain injuries at near-term ages (postmenstrual age, 36-41 weeks), such as focal abnormalities, intraventricular hemorrhage, chromosomal abnormalities, congenital infections, cystic periventricular leukomalacia, and Down syndrome. Infants with SH were distinguished from those without SH by TSH values of > 6 and < 20 μU/mL; the measurements were averaged across three serial TFT administered at 1, 3, and 6 weeks of life. Clinical characteristics included gestational age, age at scan, birth weight, body weight at MRI scan, Apgar scores at 1 and 5 min, cesarean section, patent ductus arteriosus and it with requiring surgery, prenatal and postnatal steroid, respiratory distress syndrome, culture-proven sepsis, necrotizing enterocolitis and it with requiring surgery, and bronchopulmonary dysplasia. Serial TFTs measuring both serum TSH and fT4 levels were routinely performed at 1, 3, and 6 weeks of postnatal age for all VLBW infants. Even if the initial TFT values obtained at 7 days of life were normal, we repeated the screening test at 3 weeks (from 14 to 21 days) and 6 weeks (from 42 to 49 days of life) because delayed elevations of TSH elevation is common among premature infants 6 . If the TFT values were abnormal (TSH > 6 μU/mL and/or fT4 < 0.8 ng/dL), the test was repeated after 1 or 2 weeks according to careful consideration by the pediatric endocrinologists at our hospital. www.nature.com/scientificreports/ protocol, and informed consent for participation in this study was acquired by the parents. All procedures were performed in compliance with the principles of the Declaration of Helsinki.

MRI acquisitions.
The CN and SH groups were examined at near-term ages (PMA 36-41 weeks) using whole-body 3 T MRI (Philips, Achieva, Best, Netherlands) with a 16-channel phase-array head coil during natural sleep while they were covered with a blanket to maintain body temperature. Their safety was ensured by monitoring pulse oximetry, respiratory rate, and heart rate by a trained pediatrician during the MR scans. For the DTI, three-dimensional (3D) echo-planar images with a single-shot spin-echo type were acquired with volume B0-shimming. The scan parameters of the DTI were as follows: TR = 4800 ms, TE = 75 ms, field of view (FOV) = 120 × 120 mm 2 , voxel sizes = 1. Reconstruction and quantification for fiber connectivity. The 3D-DTI images were used to reconstruct the thalamocortical connectivity based on fiber length using the FMRIB Software Library (FSL, https:// fsl. fmrib. ox. ac. uk/ fsl/ fslwi ki) 57 . First, we implemented motion correction using mcflirt, which is a part of the FSL 58 . Twenty preterm infants whose fractional anisotropy maps showed motion artifacts were removed from the DTI analysis (Fig. 5); hence, we were able to determine fiber integrity for 65 infants (CN, 39; SH, 29). Second, an early eddy-current technique provided in the FSL (eddy_correct) was applied to the DTI images to correct subject distortions and movements 59 . We obtained a parenchymal b0-map, processed it using the Brain Extraction Tool (BET) 60 from 16 directional DTI images, applied linear registration from diffusion space to a neonatal T2-weighted image of the University of North Carolina (UNC) atlases 61 , and vice versa. Subsequently, the bedpostx GPU-version 62 , which default corrects partial volume effect 63 since the slice thickness of this study was a bit thick, was executed with the advanced options (Fibers 3 and Rician option for uniform noise levels) to rapidly estimate fiber distributions for each voxel of the brain. The GPU-based probtrackx was also employed to extract information related to fiber connectivity between the thalamus and various cerebral regions 64 . The thalamus in the neonatal UNC-labels was assigned as the main seed, and each cerebral UNC region was allotted as a stop criterion to check the left and right thalamocortical integrity. The regions of interest from the thalamus to each cortex were defined by Ball et al. 30 , as illustrated in Fig. 5. Lastly, the threshold-free cluster enhancement Figure 5. An illustration of the extraction of tractography-based fiber lengths. Image datasets were chosen according to whether motion artifacts were corrected. After eddy-current correction, the probabilistic process was initiated to reconstruct fiber lengths. The seed point was set to the thalamus, and each endpoint is present in the UNC label-map (colorful regions overlaid on a gray morphology atlas). The color-map regions used to mask the image are marked in a table with each anatomical name. The number of asterisks appears the combination of several anatomical regions, which include that * is dorsal and medial superior frontal gyrus, ** is pars triangularis and pars opercularis, and *** is superior, middle, inferior, and medial orbitofrontal gyrus. Sag sagittal, Ax axial, UNC University of North Carolina atlas. www.nature.com/scientificreports/ (TFCE) 65 of the FSL was determined with randomize to find the significant thalamocortical tracts between the control and the SH. The parameters for the TFCE were designed for a two-sample unpaired t-test without not modeling mean values in the design matrix. Subsequently, the threshold value was selected with 0.95; it is equivalent to p < 0.05. We controlled family-wise error rate after TFCE.
Fiber lateralization of the thalamocortical tracts. Cerebral myelination in term-born neonates progresses rapidly in the whole brain between 6-20 months; between 21-48 months of life, myelination gradually escalates in the frontal and temporal lobes relative to the parietal and occipital lobes 66 . In addition, the cerebral regions of newborns feature neurodevelopmental asymmetries. Some regions, such as the superior temporal gyrus and orbitofrontal gyrus, develop independently to become dominant in the left or right hemisphere [46][47][48] . This extent of this difference is determined by lateralization evaluations 46 . Thus, we utilized the lateralization index and TFCE analysis to check whether the developmental processes of the significant thalamocortical tracts were changed: where Lt is the left-dependent fiber length, and Rt is the right. Positive LI values indicate left-dominant function, while negative values indicate right dominance. Lastly, each LI between the CN and the SH was averaged with standard errors and then compared to investigate the developmental asymmetries of infants with SH with their CN counterparts.

Statistical analysis for subjects and lateralization indices.
Although the significant thalamocortical tracts of the fiber lengths were extracted through the TFCE in the FSL, the differences in subject populations and cerebral asymmetries were evaluated using SPSS 22.0. The statistical analysis was conducted with the 5000 permutations and two-group differences. The analysis was adjusted for the covariates of gestational weeks and age at scan. The corresponding values for the significant UNC tracts were extracted using the cluster function of the FSL, which was calculated as average values with standard errors. The chi-squared distributions and independent t-tests were implemented for demographic and clinical characteristics. We also analyzed each LI value of the significant thalamocortical regions calculated with fiber lengths, which were evaluated by ANCOVA adjusting the gestational age and the age at scan. The comparison of the main effect was conducted with Bonferroni correction, and the adjusted p value was assigned to a significant range of < 0.05.

Data availability
The datasets generated during and/or analyzed during the current study are not publicly available due to inability to share personal information according to research ethics but are available from the corresponding author on reasonable request. Correspondence and requests for materials should be addressed to Y.H.J (ryanjang93@ hanyang.ac.kr) or H.J.L (blesslee77@hanmail.net).